Project Summary: Runx2 is a master regulator of bone formation and has two major isoforms that are functionally similar but are controlled by two distinct promoters (P1 and P2). We hypothesize that the ability of Runx2 to govern skeletal differentiation is partly based on the spatio-temporal expression patterns of each promoter. The goals of the proposed work are 1) to understand the molecular mechanisms that govern utilization of the two Runx2 promoters and thus drive osteoblast growth and differentiation and 2) to employ this knowledge to engineer bone substitutes that promote faster healing by mimicking the natural processes of the body. Our overall hypothesis is that Runx2 cross-regulation, BMP-2, homeodomain proteins, and Wnt signaling differentially affect the P1 and P2 promoters, which results in P2-related mRNA expression in early bone development and P1-related mRNA expression during late osteoblast differentiation. We have developed a mouse model where exon 1 (used only in P1 activation) is replaced by LacZ. These mice are unique in that they allow for exquisite discrimination between the use of the P1 and P2 promoters through beta-galactosidase detection and qRT-PCR. Our specific aims are 1) Delineate the precise timing for activation and expression of the bone-specific Runx2 P1 promoter and its role in supporting bone development, 2) Define the mechanisms by which the endogenous Runx2 gene locus is regulated through the P1 and P2 promoters, and 3) Determine the ability of surface-bound BMP-2-derived peptides to recapitulate BMP-2 activated osteogenesis. This work will involve histological studies of mice and dynamic ex vivo cell cultures that undergo osteogenesis and chondrogenesis. Techniques will include in situ hybridization, qRT-PCR, viral transfection, shRNA, and chromatin immunoprecipitation. [unreadable] Relevance: Runx2 has been implicated in cleidocranial dysplasia, a human disease that affects the cranial bones and clavicles. This work will define the mechanisms that regulate Runx2 promoter utilization, which in turn affects bone and cartilage differentiation. A more thorough understanding of these processes will provide insights into skeletal diseases that are a result of disruptions of the native control mechanisms. A better understanding of osteoblast differentiation will also aid in development of bone substitutes. [unreadable] [unreadable] [unreadable]